Molecular Studies on Cystic Echinococcosis of Camel (Camelus dromedarius) and Report of Echinococcus ortleppi in Iran.

BACKGROUND
Cystic echinococcosis (CE) is one of the most important zoonotic diseases; caused by different genotypes of Echinococcus spp. Camels have an important role in transmission cycle of E. granulosus especially, in desert areas. This study aimed to investigate molecular characterization of hydatid cysts isolates from one-humped camel (Camelus dromedarius) and to show its molecular and phylogenic status in this important CE host in the central part of Iran.


METHODS
Twenty hydatid cyst samples (14 fertile and 6 calcified) were collected from 56 slaughtered camels in Central part of Iran. Extraction of DNA from 14 fertile samples was achieved followed by molecular studies on two mitochondrial genes (nad1 and cox1).


RESULTS
Blast and phylogenetic analysis on sequenced genes showed the presence of G1 (28.6%), G3 (28.6%) and G6 (35.7%) genotypes in the samples. However, one sample was detected as E. ortleppi (G5) with 99% homology with G5 isolated from camel in Egypt (AB921055) and Sudan (JX912709).


CONCLUSION
Presence of E. ortleppi, originally the cattle genotype, is reported for the first time in Iran. Due to the potential of infecting human by E. ortleppi; more attention should be paid to this zoonotic genotype in this region.

E. ortleppi (G5 strain) as a common cattle strain that is geographically distributed in Europe, Africa, Southern Asia and the Americas (7,32); has been reported from camel in Sudan and Egypt (33,34). Human infection by this genotype has also been reported from several countries including Argentina, Brazil, Mexico, Netherlands, South Africa, France and India (35)(36)(37).
This study aimed to investigate molecular and phylogenetic data on hydatid cysts isolated from one-humped camel (Camelus dromedarius) in Iran.; where, G1, G3, and G6 genotypes have been reported from camel, earlier (20,24).

Materials and Methods
Twenty hydatid cysts samples including 18 (90%) cysts from lung and 2 (10%) cysts from liver of 56 slaughtered camels were collected from February to March 2015 in Najaf Abad district abattoir, Isfahan Province, Central part of Iran. The samples were immediately transferred to the laboratory in cool condition. Hydatid cyst fluids (HCF) including protoscoleces (PSCs), were collected by sterile syringes and transferred into suitable and clean falcon tubes. The tubes were centrifuged at 3000 × g. The PSCs were collected and transferred into alcohol 70% for molecular studies.
Micro-tubes containing PSCs were centrifuged at 3000 × g followed by removing their supernatants. The packed sediment (30-100 μl) was transferred into a new 1.5 micro-tube and washed three times by distilled water for removing of excess alcohol. A total of 300 μl of lysis buffer was added to each sample. Freeze and thaw procedure was applied to each tube for five times -each for 3 min-using liquid nitrogen and boiling water for surface cracking of PSCs. A total of 25 μl of proteinase K was added to each sample and incubated at 37º C overnight. The DNA was extracted by phenol-chloroform protocol. In brief, 300 μl of phenol-chloroform-isoamyl alcohol was added to each sample and centrifuged at 2000 × g for 5 minutes. The supernatant was transferred into a new micro-tube and previous step was repeated. The same volume of absolute ethanol was added to the supernatant. Sodium acetate 3M, was added as much as 0.1 of the mentioned volume and was incubated at -20º C for 30 minutes. The sample was then centrifuged at 5000 × g for 12 minutes and its supernatant was poured off. The pellet was added a total of 300 μl of ethanol 70 % and centrifuged at 2000 × g for 5 minutes. The supernatant was discarded followed by waiting for drying the remaining alcohol from the samples and finally each sample was added 50 μl of deionized water and transferred into -20° C, until use.
PCR reagents and thermal cycler program were similar in both cox1 and nad1 genes amplification. PCR reactions were applied in a final volume of 50 μl, including 2.5 μl genomic DNA, 3.5 mM MgCl2, 250 µM of dNTPs, 25 p mol. of each primer and 2 U of Taq polymerase. The following temperature profile was used for DNA amplification: 40 cycles of 94º C for 45 s, 51° C for 35 s, 72° C for 45 s, followed by a final extension at 72° C for 10 min. Positive (confirmed DNA samples) and negative (no added DNA) controls were used for each PCR program for accuracy. PCR products were visualized using electrophoresis with 1.5 % agarose gel in TAE buffer and stained with GelRed (Biotium®). A 100-bp molecular ladder was used as DNA size marker in each gel for estimating the size of the bands. Gels were observed and photographed using a UV-trans illuminator (Uvitec®).
All PCR primary products of both cox1 and nad1 genes were purified by purification kit (Vivantis®) and sequenced in two directions using the similar forward and reverse primers applied in the PCR. Sequence results were edited and aligned by Genius (40) and BioEdit (41) softwares.
To confirm the identity of the obtained sequences in comparison with the GenBank nucleotide database, all samples were blasted using NCBI (National Center for Biotechnology Information, Bethesda, MD, USA). Phylogenetic trees were constructed using Maximum Likelihood Tree implemented in MEGA software version 7 (42). Bootstrap analysis was used to evaluate the reliability of inferred trees from MEGA 7 software. Nucleotide se-quences of cox 1 and nad1 genes belonged to Taenia saginata with GenBank accession numbers AB494480 and AM503345, were used as out groups in the phylogenetic trees, respectively.

Results
Out of 20 hydatid cyst samples, 18 (90%) cysts belonged to lung and two (10%) to the liver. A total of 70% (14/20) of the cysts were fertile while, 30% (6/20) were calcified. DNA isolation from calcified cysts was negative so, they were ruled out from molecular studies. PCR-based assay with specific primers yielded two different bands of 450-bp and 470-bp in PCR of cox1 (12 samples) and nad1 (3 samples) genes, respectively. DNA isolation from fourteen fertile cysts was successful which their PCR products were sequenced. Blast analysis of the sequenced data using GenBank database, indicated the presence of G1 in 28.6% (4/14), G3 in 28.6% (4/14), G6 in 35.7% (5/14) and G5 in 7.1% (1/14) isolates in the current study. The amplified genes and the accession numbers for the detected strains are shown in Table 1. It should be considered that detected G5 genotype, using cox1 gene, subsequently, was also confirmed by amplification of nad1 gene. The partial sequences generated from cox1 and nad1 genes for G5 strain were deposited in the GenBank under the accession numbers KT988115 and KT988119, respectively.
In our G3 and G1 genotypes (obtained by cox1) there was only one alteration in nucleotide comparing to the used reference sequences: (KT074949) for G3 and (KF731903) for G1 genotypes, respectively. In five G6 samples detected by cox1 and nad1 primers in the present study, 99% homology was observed with reference sequences used in the phylogenetic trees. The G5 genotype isolated from camel in Egypt (AB921055) and Sudan (JX912709) were 99% identical to the G5 detected in the current study with only onealteration nucleotide sequences. However, G5 of the current study (cox1) had 100% homol-ogy with G5 obtained from lemur (KU378107) and spotted deer (JX068638) in the United Kingdom. The relationship between these isolates and other similar genotypes identified worldwide are shown by phylogenetic trees for cox1 (Fig.  1) and nad1 (Fig. 2) genes. Using cox1 and nad1, 18 and 12 isolates were analyzed, respectively and related phylogenetic trees were constructed ( Fig.1 and Fig.2).

Discussion
Cystic echinococcosis (CE), affects many people throughout the world, although, ad-vances in diagnosis and treatment of CE had been achieved in the recent years. There is still a limit to the disease control. As an endemic region with high incidence of CE, the disease is considered as a public health and socioeconomic problem in Iran, yet (2)(3)(4). To control this disease, strategies including surveying on different aspects of parasite should be more considered in the endemic regions (43). However, investigations on the epidemiology and different genotypes of parasites in the intermediate and final hosts should be considered in any endemic area to achieve the evidence-based control and management programs (36).  Fig. 1: Genetic relationships of obtained genotypes from camel in the present study and reference sequences related genotypes of E. granulosus as well as Taenia saginata as the out-group. The relationships were inferred based on phylogenetic tree (cox1 gene). The phylogenetic tree was constructed using Maximum Likelihood Tree implemented in MEGA software version 7

Fig. 2:
Genetic relationships of obtained genotypes from camel in the present study and reference sequences related genotypes of E. granulosus as well as Taenia saginata as the outgroup. The relation-ships were inferred based on phylogenetic tree (nad1 gene). The phylogenetic tree was constructed using Maximum Likelihood Tree implemented in MEGA software version 7 Different genotypes of E. granulosus including G1, G2, G3, G6 and G7 have been reported from different hosts in Iran (9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Camels as important intermediate hosts for CE, specially, in desert areas, have been studied in Iran and molecular studies on nuclear and mitochondrial genes have indicated the presence of G6 genotype as the dominant genotype of E. granulosus in camels (3,23,44,45); however, the presence of G1 and G3 genotypes in camel hydatid cysts have also been reported from Iran (21,24,25).
The G6 genotype has been known as common camel strain but, in some areas, G3 genotype of E. granulosus has been shown the dominant genotype in camels (24,44); however, G1 has also been considered as a noticeable genotype in camels (21,22,25). In the current study, G6 was the dominant genotype and detected in 35.7% of samples. The highest infection rate in camel has been reported from Isfahan and Khorasan Razavi and the lowest rate in Kerman and Semnan Provinces (22,46). In the present study, the lungs were the most infected organ, which is similar to previous studies (9,46,47).
In the African countries, the G6 has been reported as the dominant genotype (3,48). The genotype of all isolates from camel in Mauritania, Algeria and Sudan has been reported to be G6 (49)(50)(51). However, other studies in Kenya and Libya have shown a noticeable prevalence of G1 strains in camel isolates (51,52). The G5 genotype, its host and distribution is different in the world. Investigation of 638 fertile cysts of cattle has shown the presence of G1 (56.6%) and G5 (43.4%) genotypes in Brazil, while the G5 was mostly isolated from lungs (53). The G5 genotype has been isolated from cattle cysts in Argentina and Italy (54,55) and in spotted deer from UK, too (56).
Existence of G5 strain has already been reported from camel in Sudan and Egypt (33,34). This genotype has also been reported from human in Argentina, Brazil, Mexico, Netherlands, South Africa, India and France (7,35,37), which makes it as an important genotype in the view of public health. The G5 genotype isolated from camel in Egypt (AB921055) and Sudan (JX912709) were 99% identical to the G5 detected in the current study with only one alteration in nucleotide sequences and similar to other studies in G5 genotypes in camel. However, G5 of the current study (cox1) had 100% homology with G5 obtained from lemur (KU378107) and spotted deer (JX068638) in the United Kingdom (56).
The camel as a natural intermediate host for E. granulosus plays an important role in the maintaining of the parasite in the nature especially in desert areas (43). On the other hand scattered camels in desert and semi desert areas, where other ruminants and carnivorous animals may live in Iran' could be infected with this important genotype.

Conclusion
As far as our knowledge, the present study genetically showed the presence E. ortleppi (G5) in camel for the first time in Iran. However, other zoonotic genotypes including the G1, G3 and G6, which have been reported from camel, were also detected in the present study. Due to the transmission potential of G5 strain to human, the finding of E. ortleppi, in camel should be more noticed in Iran. The distribution of G5 genotype in Iran is not known. However, more studies are needed to find the distribution of G5 genotype in Iran. More molecular studies on cattle and camel hydatid cysts are needed to find the main reservoir of E. ortleppi in Iran. Moreover, molecular and parasitological studies on different final hosts will evaluate the probable existence and its circulation in this region.